The topography of the active sites of rhesus carbonic anhydrases (CA) I and II, rabbit CA I, sheep CA II, and dog CA I has been studied with the aid of spin-labeled analogs of acetazolamide and sulfanilamide. Electron spin resonance measurements indicated that the pyrrolidine ring of 2,2,5,5-tetramethyl-3-(Rho-sulfamoylphenyl) carbamoyl)-1-pyrrolidinyloxyl became highly immobilized when this label bound to the active site of rhesus CA I. As the chain length between the aromatic and pyrrolidine rings was increased, the mobility of the nitroxide group of the enzyme-bound inhibitor progressively increased until with 2,2,5,5-tetramethyl-3-(Rho-sulfamoylphenyl) carbamoyl)-methyl)carbamoyl)-1-pyrrolidinyloxyl there was only minimal interaction between the heterocyclic ring and the active site of rhesus CA I. These findings suggest that the active site of rhesus CA I is a cleft about 14 A deep. Similar experiments indicated that the active sites of rhesus CA II, dog CA I and sheep CA II were similar in size to that of rhesus CA I, while the active site of rabbit CA was somewhat deeper. Spin-labeled inhibitor 2,2,6,6-tetramethyl-4-(Rho-sulfamoylbenzamide) piperidinooxyl became highly immobilized on binding to type II (high activity) CA but exhibited isotropic motion at the active sites of type II (high activity) CA but exhibited isotropic motion at the active sites of type II (high activity) CA but exhibited isotropic motion at the active sites of type I (low activity) isozymes. An attempt has been made to explain these results in terms of the three-dimensional structure of the active sites of human CA I and II.